Xenopus fibrillin is expressed in the organizer and is the earliest component of matrix at the developing notochord-somite boundary.

We identify a Xenopus fibrillin homolog (XF), and show that its earliest developmental expression is in presumptive dorsal mesoderm at gastrulation, and that XF expression is regulated by mesoderm-inducing factors in animal cap assays. XF protein is also first detected in presumptive mesoderm, but is concentrated specifically into extracellular-matrix structures that begin to develop de novo by mid-gastrulation at both of the bilateral presumptive notochord-somite boundaries. Later in embryogenesis, XF protein is localized to the extracellular matrix at tissue boundaries, where it is found surrounding the notochord, the somites, and the neural tube, as well as under the epidermis. This pattern of protein deposition combines to give the appearance of an "embryonic skeleton," suggesting that one role for XF is to serve as a mechanical element in the embryo prior to bone deposition.

The role of combinational coding by homeodomain and bHLH transcription factors in retinal cell fate specification.

Two major families of transcription factors (TFs), basic helix-loop-helix (bHLH) and homeodomain (HD), are known to be involved in cell fate identity. Some recent findings suggest that these TFs are used combinatorially to code for cellular determination in the retina. However, neither the extent nor the efficiency of such a combinatorial coding mechanism has been tested. To look systematically for interactions between these two TF types that would address these questions, we used a matrix analysis. We co-expressed each of six retinally expressed bHLH TFs (XNeuroD; XNgnr-1; Xath3; Xath5; Xash1; Xash3) with each of eight retinally expressed HD TFs (XRx1; XOptx2; XSix3; XPax6; XOtx2; XOtx5b; XBH; XChx10) in retinal progenitors of Xenopus laevis using targeted lipofection. The effects of each of these combinations were assayed on the six major cell types in the retina: Retinal ganglion cells (GCs), Amacrines (ACs), Bipolars (BCs), Horizontals (HCs), Photoreceptors (PRs), and Muller cells (MCs), creating 288 result categories. Multiple-way ANOVA indicated that in 14 categories, there were interactions between the two TFs that produced significantly more or less of a particular cell type than either of the components alone. However, even the most effective combinations were incapable of generating more than 65% of any particular cell type. We therefore used the same techniques to misexpress selected combinations of three TFs in retinal progenitors, but found no further enhancements of particular cell fates, indicating that other factors are probably involved in cell type specification. To test whether particular combinations were essential for horizontal fates, we made VP16 and EnR fusion constructs of some of the factors to provide dominant negative transcriptional activities. Our results confirmed that normal activities of certain combinations were sufficient, and that individually these activities were important for this fate.

The zebrafish as a tool for understanding the biology of visual disorders.

Retinal degenerations are the commonest cause of blindness in the Western world, affecting 5% of the population, yet remain largely untreatable. A better understanding of the mechanisms of disease is needed. Zebrafish fill a gap in the current repertoire of models, offering genetic tractability in a vertebrate. Their retina has many similarities with a human retina. Importantly, unlike rodents, they have rich colour vision, offering the potential to model the macular degenerations. A variety of physiological assays, genetic manipulations and histological tools have been developed and useful models of human disease created.

Temporal coordinates: the genes that fix cell fate with birth order.

Drosophila neuroblasts sequentially express a set of four transcription factors that specify the fates of their progeny according to the successive order of their generation. In analogy to spatial coordinate genes that specify neuroblasts by position, these sequentially expressed genes can be called "temporal coordinate genes."

Cellular competence plays a role in photoreceptor differentiation in the developing Xenopus retina.

Factors in the environment appear to be responsible for inducing many of the cell fates in the retina, including, for example, photoreceptors. Further, there is a conserved order of histogenesis in the vertebrate retina, suggesting that a temporal mechanism interacts in the control of cellular determination. The temporal mechanism involved could result from different inducing signals being released at different times. Alternatively, the inducing signals might be present at many stages, but an autonomous clock could regulate the competence of cells to respond to them. To differentiate between these mechanisms, cells from young embryonic retinas were dissociated and grown together with those from older embryos, and the timing of photoreceptor determination assayed. Young cells appeared uninfluenced by older cells, expressing photoreceptor markers on the same time schedule as when cultured alone. A similar result was obtained when the heterochronic mixing was done in vivo by grafting a small plug of optic vesicle from younger embryos into older hosts. Even the graft cells at the immediate margin of the transplant failed to express photoreceptor markers earlier than normal, despite their being in contact with older, strongly expressing host cells. We conclude that retinal progenitors intrinsically acquire the ability to respond to photoreceptor-inducing cues by a mechanism that runs on a cell autonomous schedule, and that the conserved order of histogenesis is based in part on this competence clock.

Semaphorin 3A elicits stage-dependent collapse, turning, and branching in Xenopus retinal growth cones.

The semaphorin receptor, neuropilin-1 (NP-1), was first identified in Xenopus as the A5 antigen and is expressed abundantly in developing retinal ganglion cells (RGCs). Here we show that growth cones acquire responsiveness to semaphorin 3A (Sema 3A) with age and that the onset of responsiveness correlates with the appearance of NP-1 immunoreactivity. Growth cones from "old" (stage 35/36) retinal explants collapse rapidly (5-10 min) in response to Sema 3A and turn away from a gradient of Sema 3A, whereas "young" growth cones (stage 24) are insensitive to Sema 3A. Moreover, transfection of full-length NP-1 into young neurons confers premature Sema 3A sensitivity. When young neurons are aged in culture they develop Sema 3A sensitivity in parallel with those in vivo, suggesting that an intrinsic mechanism of NP-1 regulation mediates this age-dependent change. Sema 3A-induced collapse is transient, and after recovery approximately 30% of growth cones extend new branches within 1 hr, implicating Sema 3A as a branching factor. Pharmacological inhibitors were used to investigate whether these three Sema 3A-induced behaviors (collapse, turning, and branching) use distinct second messenger signaling pathways. All three behaviors were found to be mediated via cGMP. In situ hybridization shows that Sema 3A is expressed in the tectum and at the anterior boundary of the optic tract where axons bend caudally, suggesting that Sema 3A/NP-1 interactions play a role in guiding axons in the optic tract and in stimulating terminal branching in the tectum.

Changing patterns of hospitalization in eating disorder patients.

OBJECTIVE: This study investigated the changing patterns of hospitalization of eating disorder patients over the past 15 years. METHOD: The records of 1,185 eating disorder patients between 1984 and 1998 were examined on several variables. RESULTS: Over the 15 years, the number of first admissions increased from 20 to 182. There was a concomitant decrease in length of stay from 149.5 days in 1984 to 23.7 days in 1998. Readmissions increased markedly from 0% during the first year to 27% of total admissions in 1998. The discharge weight of anorectic patients significantly decreased from a body mass index (BMI) of 19.3 in 1984 to 17.7 in 1998. These changes were particularly salient in the past 3 years, concurrent with a dramatic rise in managed care cases. CONCLUSIONS: Over the past 15 years, eating disorder hospital treatment has metamorphozed from long-term treatment of a disorder to stabilization of acute episodes. For some patients, this change has been deleterious and not cost effective.

Cell cycle and cell fate in the nervous system.

Recently, a number of molecules originally thought to have a primary role in cell determination have been shown to affect the cell cycle at specific check points, while other molecules discovered for their roles in the cell cycle progression are known to affect the determination and differentiation of neurons. These discoveries have led to a more detailed investigation of the complex molecular machinery that co-ordinates proliferation and differentiation.

Seminal plasma trace metal levels in industrial workers.

This study compares the seminal plasma trace metal levels of hospital workers with groups of industrial workers in a petroleum refinery, smelter, and chemical plant. The metals measured were the essential metals (copper, zinc, nickel, cobalt, and manganese) and the toxic metals (lead, cadmium, and aluminum). The group mean +/- SE metal level for each group (50 subjects per group) was calculated, and the statistical significance of the group mean differences of the industrial groups with the hospital group (control) was determined by the Student's t-test. The differences observed in the smelter group were increased copper and zinc (p < or = 0.001) and decreased nickel, cobalt, and manganese (p < or = 0.001, < or = 0.01). The refinery group differences were increased copper, zinc, and nickel (p < or = 0.001) but decreased cobalt and manganese (p < or = 0.001). The chemical group differences were increased zinc (p < or = 0.001) and decreased cobalt (p < or = 0.001). The seminal plasma levels of the toxic metals lead and aluminum were increased in each of the industrial groups (p < or = 0.001). Concurrent differences were (1) decreased accumulation of nickel, cobalt, and manganese in the smelter group, (2) decreased cobalt and manganese in the refinery group, and (3) only decreased cobalt in the chemical group.

Retinal stem cells in vertebrates.

In fish and amphibia, retinal stem cells located in the periphery of the retina, the ciliary marginal zone (CMZ), produce new neurons in the retina throughout life. In these species, the retina grows to keep pace with the enlarging body. When birds or mammals reach adult proportions, however, their retinas stop growing so there appears to be no need for such a proliferative area with stem cells. It is a surprise, therefore, that recent data suggest that a region similar to the CMZ of fish and amphibia exists in the postnatal chick and the adult mouse.

The multiple decisions made by growth cones of RGCs as they navigate from the retina to the tectum in Xenopus embryos.

Retinal ganglion cells (RGCs) of Xenopus laevis send axons along a stereospecific pathway from the retina to their target the optic tectum. Viewed from the point of the growth cone, this journey is reflected by discrete processes of axon initiation, axon outgrowth, navigation, target recognition, and innervation. These processes are characterised by distinct signalling mechanisms that trigger dynamic changes in growth cone morphology and behavior. Here we review work primarily from our laboratory, examining these events from a cellular and molecular perspective, focusing on the roles of FGFs, netrins, receptors, and intracellular effectors.

Ephrin-B regulates the Ipsilateral routing of retinal axons at the optic chiasm.

In Xenopus tadpoles, all retinal ganglion cells (RGCs) send axons contralaterally across the optic chiasm. At metamorphosis, a subpopulation of EphB-expressing RGCs in the ventrotemporal retina begin to project ipsilaterally. However, when these metamorphic RGCs are grafted into embryos, they project contralaterally, suggesting that the embryonic chiasm lacks signals that guide axons ipsilaterally. Ephrin-B is expressed discretely at the chiasm of metamorphic but not premetamorphic Xenopus. When expressed prematurely in the embryonic chiasm, ephrin-B causes precocious ipsilateral projections from the EphB-expressing RGCs. Ephrin-B is also found in the chiasm of mammals, which have ipsilateral projections, but not in the chiasm of fish and birds, which do not. These results suggest that ephrin-B/EphB interactions play a key role in the sorting of axons at the vertebrate chiasm.

Determination of vertebrate retinal progenitor cell fate by the Notch pathway and basic helix-loop-helix transcription factors.

The retina is an excellent system in which to study neural cell fate decision mechanisms. It is an organized laminated structure with a limited array of cell types. During the last 5 years, experiments that perturb normal gene expression have highlighted some molecular mechanisms involved in cellular fate choice in the retina. By controlling when a retinoblast is allowed to differentiate, Delta-Notch signaling plays a critical role in the generation of neuronal diversity in the vertebrate retina. When cells are released from the inhibition mediated by the Delta-Notch pathway, basic helix-loop-helix (bHLH) transcription factors act as intrinsic factors that bias neuroblasts towards particular fates. In this review, we present an overview of the data leading to these conclusions on the role of the Delta-Notch pathway and the bHLH proteins on cell fate decisions during vertebrate retinogenesis.

p27Xic1, a Cdk inhibitor, promotes the determination of glial cells in Xenopus retina.

p27Xic1, a member of the Cip/Kip family of Cdk inhibitors, besides its known function of inhibiting cell division, induces Müller glia from retinoblasts. This novel gliogenic function of p27Xic1 is mediated by part of the N-terminal domain near but distinct from the region that inhibits cyclin-dependent kinases. Cotransfections with dominant-negative and constitutively active Delta and Notch constructs indicate that the gliogenic effects of p27Xic1 work within the context of an active Notch pathway. The gradual increase of p27Xic1 in the developing retina thus not only limits the number of retinal cells but also increasingly favors the fate of the last cell type to be born in the retina, the Müller glia.

X-ngnr-1 and Xath3 promote ectopic expression of sensory neuron markers in the neurula ectoderm and have distinct inducing properties in the retina.

Xath3 encodes a Xenopus neuronal-specific basic helix-loop-helix transcription factor related to the Drosophila proneural factor atonal. We show here that Xath3 acts downstream of X-ngnr-1 during neuronal differentiation in the neural plate and retina and that its expression and activity are modulated by Notch signaling. X-ngnr-1 activates Xath3 and NeuroD by different mechanisms, and the latter two genes crossactivate each other. In the ectoderm, X-ngnr-1 and Xath3 have similar activities, inducing ectopic sensory neurons. Among the sensory-specific markers tested, only those that label cranial neurons were found to be ectopically activated. By contrast, in the retina, X-ngnr-1 and Xath3 overexpression promote the development of overlapping but distinct subtypes of retinal neurons. Together, these data suggest that X-ngnr-1 and Xath3 regulate successive stages of early neuronal differentiation and that, in addition to their general proneural properties, they may contribute, in a context-dependent manner, to some aspect of neuronal identity.

Amniotic fluid B12, calcium, and lead levels associated with neural tube defects.

The group mean differences and relationships between folate, B12, calcium, lead, and methionine levels in amniotic fluid from 29 non-NTD and 11 NTD pregnancies (15-20 weeks' gestation age) were determined. The study population was predominantly Hispanic in both groups (48% in non-NTD and 36% in NTD group) with an average maternal age of 29 years in the non-NTD group and 24 years in the NTD group; and, an average gestation age of 18.5 weeks in the non-NTD and 17.5 weeks in the NTD group. The folate, B12, lead, calcium, and methionine levels of the two groups were compared by Student's t-test and by Pearson's correlation. The NTD levels were lower for calcium (p< or =0.0001), B12 (p< or =0.001), and methionine (P< or =0.001); but, the lead level was higher (p< or =0.0001). A negative correlation was observed between lead and both B12 (p< or =0.007) and methionine (p< or =0.02).

The effect of ascorbic acid supplementation on the nicotine metabolism of smokers.

BACKGROUND: The relationship of serum ascorbic acid (AA) levels and excretion of nicotine metabolites was determined in 75 men who smoked at least one pack of cigarettes per day. METHODS: The subjects were randomly divided into three groups of 25 each who received a placebo, 200 mg of supplementation, or 1000 mg of supplementation of AA per day for 1 month. Baseline and weekly serum AA levels were determined and simultaneous estimates of urinary excretion of nicotine metabolites as cotinine equivalents (CE). RESULTS: The group mean serum AA levels in the placebo group decreased 13% after 2 weeks; the group mean serum levels of the supplemented groups increased significantly after 1 week (P

The neuronal architecture of Xenopus retinal ganglion cells is sculpted by rho-family GTPases in vivo.

Dendritogenesis, axonogenesis, pathfinding, and target recognition are all affected in distinct ways when Xenopus retinal ganglion cells (RGCs) are transfected with constitutively active (ca), wild-type (wt), and dominant negative (dn) Rho-family GTPases in vivo. Dendritogenesis required Rac1 and Cdc42 activity. Moreover, ca-Rac1 caused dendrite hyperproliferation. Axonogenesis, in contrast, was inhibited by ca-Rac1. This phenotype was partially rescued by the coexpression of dn cyclin-dependent kinase (Cdk5), a proposed effector of Rac1, suggesting that Rac1 activity must be regulated tightly for normal axonogenesis. Growth cone morphology was particularly sensitive to dn-RhoA and wt-Cdc42 constructs. These also caused targeting errors, such as tectal bypass, suggesting that cytoskeletal rearrangements are involved in target recognition and are transduced by these pathways.